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BACKGROUND: Mechanisms underlying the pro gression of diabetic kidney disease to ESKD are not fully understood. METHODS: We performed global microRNA (miRNA) analysis on plasma from two cohorts consisting of 375 individuals with type 1 and type 2 diabetes with late diabetic kidney disease, and targeted proteomics analysis on plasma from four cohorts consisting of 746 individuals with late and early diabetic kidney disease. We examined structural lesions in kidney biopsy specimens from the 105 individuals with early diabetic kidney disease. Human umbilical vein endothelial cells were used to assess the effects of miRNA mimics or inhibitors on regulation of candidate proteins. RESULTS: In the late diabetic kidney disease cohorts, we identified 17 circulating miRNAs, represented by four exemplars (miR-1287-5p, miR-197-5p, miR-339-5p, and miR-328-3p), that were strongly associated with 10-year risk of ESKD. These miRNAs targeted proteins in the axon guidance pathway. Circulating levels of six of these proteins—most notably, EFNA4 and EPHA2—were strongly associated with 10-year risk of ESKD in all cohorts. Furthermore, circulating levels of these proteins correlated with severity of structural lesions in kidney biopsy specimens. In contrast, expression levels of genes encoding these proteins had no apparent effects on the lesions. In in vitro experiments, mimics of miR-1287-5p and miR-197-5p and inhibitors of miR-339-5p and miR-328-3p upregulated concentrations of EPHA2 in either cell lysate, supernatant, or both. CONCLUSIONS: This study reveals novel mechanisms involved in progression to ESKD and points to the importance of systemic factors in the development of diabetic kidney disease. Some circulating miRNAs and axon guidance pathway proteins represent potential targets for new therapies to prevent and treat this condition.

Diabetic kidney disease (DKD) and its major clinical manifestation, progressive renal decline that leads to end-stage renal disease (ESRD), are a major health burden for individuals with diabetes. The disease process that underlies progressive renal decline comprises factors that increase risk as well as factors that protect against this outcome. Using untargeted proteomic profiling of circulating proteins from individuals in two independent cohorts with type 1 and type 2 diabetes and varying stages of DKD followed for 7 to 15 years, we identified three elevated plasma proteins-fibroblast growth factor 20 (OR, 0.69; 95% CI, 0.54 to 0.88), angiopoietin-1 (OR, 0.72; 95% CI, 0.57 to 0.91), and tumor necrosis factor ligand superfamily member 12 (OR, 0.75; 95% CI, 0.59 to 0.95)-that were associated with protection against progressive renal decline and progression to ESRD. The combined effect of these three protective proteins was demonstrated by very low cumulative risk of ESRD in those who had baseline concentrations above median for all three proteins, whereas the cumulative risk of ESRD was high in those with concentrations below median for these proteins at the beginning of follow-up. This protective effect was shown to be independent from circulating inflammatory proteins and clinical covariates and was confirmed in a third cohort of diabetic individuals with normal renal function. These three protective proteins may serve as biomarkers to stratify diabetic individuals according to risk of progression to ESRD and might also be investigated as potential therapeutics to delay or prevent the onset of ESRD.

We recently identified a kidney risk inflammatory signature (KRIS), comprising 6 TNF receptors (including TNFR1 and TNFR2) and 11 inflammatory proteins. Elevated levels of these proteins in circulation were strongly associated with risk of the development of end-stage kidney disease (ESKD) during 10-year follow-up. It has been hypothesized that elevated levels of these proteins in circulation might reflect (be markers of) systemic exposure to TNFα. In this in vitro study, we examined intracellular and extracellular levels of these proteins in human umbilical vein endothelial cells (HUVECs) exposed to TNFα in the presence of hyperglycemia. KRIS proteins as well as 1300 other proteins were measured using the SOMAscan proteomics platform. Four KRIS proteins (including TNFR1) were down-regulated and only 1 protein (IL18R1) was up-regulated in the extracellular fraction of TNFα-stimulated HUVECs. In the intracellular fraction, one KRIS protein was down-regulated (CCL14) and 1 protein was up-regulated (IL18R1). The levels of other KRIS proteins were not affected by exposure to TNFα. HUVECs exposed to a hyperglycemic and inflammatory environment also showed significant up-regulation of a distinct set of 53 proteins (mainly in extracellular fraction). In our previous study, circulating levels of these proteins were not associated with progression to ESKD in diabetes.

This study comprehensively evaluated the association between known circulating tumor necrosis factor (TNF) superfamily ligands and receptors and the development of early progressive kidney decline (PKD) leading to end-stage kidney disease (ESKD) in Type 1 diabetes. Participants for the study were from the Macro-Albuminuria Study (198 individuals), and the Micro-Albuminuria Study (148 individuals) of the Joslin Kidney Study. All individuals initially had normal kidney function and were followed for seven-fifteen years to determine the slope of the estimate glomerular filtration rate and to ascertain onset of ESKD. Plasma concentrations of 25 TNF superfamily proteins were measured using proximity extension assay applied in the OLINK proteomics platform. In the both studies risk of early PKD, determined as estimated glomerular filtration rate loss greater than or equal to three ml/min/1.73m2/year, was associated with elevated circulating levels of 13 of 19 TNF receptors examined. In the Macro-Albuminuria Study, we obtained similar findings for risk of progression to ESKD. These receptors comprised: TNF-R1A, -R1B, -R3, -R4, -R6, -R6B, -R7, -R10A, -R10B, -R11A, -R14, -R21, and -R27. Serial measurements showed that circulating levels of these TNF receptors had increased before the onset of PKD. In contrast, none of the six measured TNF ligands showed association with risk of early PKD. Of significance, the disease process that underlies PKD leading to ESKD in Type 1 diabetes has a profile also seen in autoimmune disorders. The mechanisms of this enrichment may be causally related to the development of PKD in Type 1 diabetes and must be investigated further. Thus, some of these receptors may be used as new risk predictors of ESKD.

SUMMARYAndrogen deprivation therapy (ADT) induces regression of recurrent and advanced prostate cancer (PrCa), but many tumors recur. To understand the response to ADT, changes in tumor volume were imaged after castration of murine PrCa models. While mouse (non-tumor) prostate begins to regress within two days of castration, murine PrCa regresses after a delay of 3-14 days in two distinct mouse models. Intra-tumoral androgens are undetectable after castration, but tumor cells proliferate during this period. Intratumoral glucocorticoids and glucocorticoid receptor (GR) protein increase, as does GR mRNA and a set of GR-regulated genes specifically in tumor epithelial cells identified using scRNAseq. A selective GR antagonist (CORT125281, relacorilant), in clinical trials for late-state PrCa, eliminates the delayed regression phenotype in both models. Thus, activated GR signaling and murine tumor proliferation following castration resembles the GR-dependent escape mechanism of castrate resistant PrCa. These results suggest simultaneous inhibition of GR and androgen receptor signaling could improve PrCa therapy.In briefAndrogen deprivation therapy for high risk and recurrent prostate cancers is initially effective, but ultimately fails; better understanding the mechanisms should improve therapy. In two murine prostate cancer models, GR signaling is activated immediately following castration, substituting for the acute reduction in AR signaling, and allowing for continued tumor growth. This continued growth is blocked by relacorilant, selective GR antagonist in clinical trials for late-state PrCa.HighlightsAndrogen deprivation therapy induces regression of prostate cancer, but tumors recurMurine PrCa continues to proliferate for 3-14 days in two distinct mouse prostate cancer modelsTumor cells proliferate during this period, and intratumoral glucocorticoids and glucocorticoid receptor (GR) protein increase, as does GR mRNA and a set of GR-regulated genesRelacorilant, a selective GR antagonist in clinical trials for late-state PrCa, eliminates the delayed regression

Background Rare variants in gene coding regions likely have a greater impact on disease-related phenotypes than common variants through disruption of their encoded protein. We searched for rare variants associated with onset of end stage kidney disease (ESKD) in individuals with type 1 diabetes at advanced kidney disease stage. Methods Gene-based exome array analysis of 15,449 genes in 5 large incidence cohorts of individuals with type 1 diabetes and proteinuria were analyzed for survival time-to-ESKD, testing the top gene in a 6th cohort (N=2,372/1,115 events all cohorts) and replicating in two retrospective case-control studies (N=1,072 cases, 752 controls). Deep resequencing of the top associated gene in 5 cohorts confirmed the findings. We performed immunohistochemistry and gene expression experiments in human control and diseased cells, and in mouse ischemia reperfusion and aristolochic acid nephropathy models. Results Protein coding variants in the hydroxysteroid 17-beta dehydrogenase 14 gene (HSD17B14), predicted to affect protein structure, had a net protective effect against development of ESKD at exome-wide significance (N=4,196; p-value=3.3x10-7). The HSD17B14 gene and encoded enzyme were robustly expressed in healthy human kidney, maximally in proximal tubular cells. Paradoxically, gene and protein expression were attenuated in human diabetic proximal tubules and in mouse kidney injury models. Expressed HSD17B14 gene and protein levels remained low without recovery after 21 days in a murine ischemic reperfusion injury model. Decreased gene expression was found in other chronic kidney disease-associated renal pathologies. Conclusions HSD17B14 gene is mechanistically involved in diabetic kidney disease. The encoded sex steroid enzyme is a druggable target, potentially opening a new avenue for therapeutic development.

The incidence of renal cell carcinoma (RCC) is increasing worldwide with an approximate 20% mortality rate. The challenge in RCC is the therapy-resistance. Cancer resistance to treatment employs multiple mechanisms due to cancer heterogeneity with multiple genetic and epigenetic alterations. These changes include aberrant overexpression of (1) anticancer cell death proteins (e.g., survivin/BIRC5), (2) DNA repair regulators (e.g., ERCC6) and (3) efflux pump proteins (e.g., ABCG2/BCRP); mutations and/or deregulation of key (4) oncogenes (e.g., MDM2, KRAS) and/or (5) tumor suppressor genes (e.g., TP5/p53); and (6) deregulation of redox-sensitive regulators (e.g., HIF, NRF2). Foci of tumor cells that have these genetic alterations and/or deregulation possess survival advantages and are selected for survival during treatment. We will review the significance of survivin (BIRC5), XIAP, MCL-1, HIF1α, HIF2α, NRF2, MDM2, MDM4, TP5/p53, KRAS and AKT in treatment resistance as the potential therapeutic biomarkers and/or targets in RCC in parallel with our analized RCC-relevant TCGA genetic results from each of these gene/protein molecules. We then present our data to show the anticancer drug FL118 modulation of these protein targets and RCC cell/tumor growth. Finally, we include additional data to show a promising FL118 analogue (FL496) for treating the specialized type 2 papillary RCC. Supplementary Information The online version contains supplementary material available at 10.1186/s13046-021-02026-1.

Background Characterization of prediagnostic Parkinson’s Disease (PD) and early prediction of subsequent development are critical for preventive interventions, risk stratification and understanding of disease pathology. This study aims to characterize the role of the prediagnostic period in PD and, using selected features from this period as novel interception points, construct a prediction model to accelerate the diagnosis in a real-world setting. Methods We constructed two sets of machine learning models: a retrospective approach highlighting exposures up to 5 years prior to PD diagnosis, and an alternative model that prospectively predicted future PD diagnosis from all individuals at their first diagnosis of a gait or tremor disorder, these being features that appeared to represent the initiation of a differential diagnostic window. Results We found many novel features captured by the retrospective models; however, the high accuracy was primarily driven from surrogate diagnoses for PD, such as gait and tremor disorders, suggesting the presence of a distinctive differential diagnostic period when the clinician already suspected PD. The model utilizing a gait/tremor diagnosis as the interception point, achieved a validation AUC of 0.874 with potential time compression to a future PD diagnosis of more than 300 days. Comparisons of predictive diagnoses between the prospective and prediagnostic cohorts suggest the presence of distinctive trajectories of PD progression based on comorbidity profiles. Conclusions Overall, our machine learning approach allows for both guiding clinical decisions such as the initiation of neuroprotective interventions and importantly, the possibility of earlier diagnosis for clinical trials for disease modifying therapies.

Rare variants in MAGEC3, members of the melanoma antigen gene family, are associated with BRCA-independent early onset ovarian cancers, while somatic mutations of this gene have been associated with kidney cancers. In this report, we quantified normal and tumor protein expression of MAGEC3 via immunohistochemistry in N=394 ovarian cancers and N=220 renal cell carcinomas. MAGEC3 protein levels fell into two categories – normal MAGEC3 and MAGEC3 loss – characterized by expression equivalent to normal tissue or significantly lower than normal tissue, respectively. Interestingly, cases with MAGEC3 loss demonstrated better overall survival in both ovarian cancers and renal cell carcinomas, which resembles patient outcomes with BRCA2 loss. MAGEC3 protein expression was associated with upregulation of pathways regulating G2/M checkpoint (NES: 4.13, FDRSTATEMENT OF TRANSLATIONAL RELEVANCEMAGEC3 protein is expressed in multiple tissues and is dysregulated in cancer. In this work, we show that ovarian and kidney cancer patients with loss of MAGEC3 protein have favorable prognosis, indicating that MAGEC3 protein level may be used as a prognostic biomarker. Integrative genomic analysis of patients spanning more than nine cancer types showed an association between MAGEC3 protein and genes affecting stress response, including those involved in cell cycle and DNA damage repair. Additionally, it is correlated with tumor mutational burden in patients with mutated oncogenes. These associations suggest that MAGEC3 protein levels may be used to identify patients with deficient DNA damage repair mechanisms that can be targeted by PARP inhibitors. To operationalize this idea, we use machine learning to predict MAGEC3 protein levels from RNA sequencing data which can facilitate the identification of patients for treatment stratification according to their MAGEC3 status.

OBJECTIVE: The role of fibrosis in early progressive renal decline in type 2 diabetes is unknown. Circulating WFDC2 (WAP four-disulfide core domain protein 2) and matrix metalloproteinase 7 (MMP-7; Matrilysin) are postulated to be biomarkers of renal fibrosis. This study examined an association of circulating levels of these proteins with early progressive renal decline. RESEARCH DESIGN AND METHODS: Individuals with type 2 diabetes enrolled in the Joslin Kidney Study with an estimated glomerular filtration rate (eGFR) ≥60 mL/min/1.73 m(2) were monitored for 6–12 years to ascertain fast early progressive renal decline, defined as eGFR loss ≥5 mL/min/1.73 m(2)/year. RESULTS: A total of 1,181 individuals were studied: 681 without and 500 with albuminuria. Median eGFR and albumin-to-creatinine ratio (ACR) at baseline were 97 mL/min/1.73 m(2) and 24 mg/g, respectively. During follow-up, 152 individuals experienced fast early progressive renal decline: 6.9% in those with normoalbuminuria and 21% with albuminuria. In both subgroups, the risk of renal decline increased with increasing baseline levels of WFDC2 (P < 0.0001) and MMP-7 (P < 0.0001). After adjustment for relevant clinical characteristics and known biomarkers, an increase by one quartile in the fibrosis index (combination of levels of WFDC2 and MMP-7) was associated with higher risk of renal decline (odds ratio 1.63; 95% CI 1.30–2.04). The association was similar and statistically significant among patients with and without albuminuria. CONCLUSIONS: Elevation of circulating profibrotic proteins is associated with the development of early progressive renal decline in type 2 diabetes. This association is independent from albuminuria status and points to the importance of the fibrotic process in the development of early renal decline.

OBJECTIVE The role of fibrosis in early progressive renal decline in type 2 diabetes is unknown. Circulating WFDC2 (WAP four-disulfide core domain protein 2) and matrix metalloproteinase 7 (MMP-7; Matrilysin) are postulated to be biomarkers of renal fibrosis. This study examined an association of circulating levels of these proteins with early progressive renal decline. RESEARCH DESIGN AND METHODS Individuals with type 2 diabetes enrolled in the Joslin Kidney Study with an estimated glomerular filtration rate (eGFR) ≥60 mL/min/1.73 m2 were monitored for 6–12 years to ascertain fast early progressive renal decline, defined as eGFR loss ≥5 mL/min/1.73 m2/year. RESULTS A total of 1,181 individuals were studied: 681 without and 500 with albuminuria. Median eGFR and albumin-to-creatinine ratio (ACR) at baseline were 97 mL/min/1.73 m2 and 24 mg/g, respectively. During follow-up, 152 individuals experienced fast early progressive renal decline: 6.9% in those with normoalbuminuria and 21% with albuminuria. In both subgroups, the risk of renal decline increased with increasing baseline levels of WFDC2 (P < 0.0001) and MMP-7 (P < 0.0001). After adjustment for relevant clinical characteristics and known biomarkers, an increase by one quartile in the fibrosis index (combination of levels of WFDC2 and MMP-7) was associated with higher risk of renal decline (odds ratio 1.63; 95% CI 1.30–2.04). The association was similar and statistically significant among patients with and without albuminuria. CONCLUSIONS Elevation of circulating profibrotic proteins is associated with the development of early progressive renal decline in type 2 diabetes. This association is independent from albuminuria status and points to the importance of the fibrotic process in the development of early renal decline.

Significance The interplay between the anorexigenic and orexigenic neurons in the arcuate nucleus that contributes to the control of feeding remains elusive. Using optogenetic stimulation, we show that activation of POMC neurons rapidly inhibits feeding behavior in fasted animals. However, simultaneous stimulation of both POMC neurons and a subset of the orexigenic neurons that express AgRP is sufficient to reverse that inhibition and trigger intense feeding behavior. We used 3D imaging and functional studies to illuminate the anatomical underpinning of both the inhibitory and excitatory events. Our work suggests that translational applications that aim to control appetite need to target the activation rather than the inhibition mechanisms., Two classes of peptide-producing neurons in the arcuate nucleus (Arc) of the hypothalamus are known to exert opposing actions on feeding: the anorexigenic neurons that express proopiomelanocortin (POMC) and the orexigenic neurons that express agouti-related protein (AgRP) and neuropeptide Y (NPY). These neurons are thought to arise from a common embryonic progenitor, but our anatomical and functional understanding of the interplay of these two peptidergic systems that contribute to the control of feeding remains incomplete. The present study uses a combination of optogenetic stimulation with viral and transgenic approaches, coupled with neural activity mapping and brain transparency visualization to demonstrate the following: (i) selective activation of Arc POMC neurons inhibits food consumption rapidly in unsated animals; (ii) activation of Arc neurons arising from POMC-expressing progenitors, including POMC and a subset of AgRP neurons, triggers robust feeding behavior, even in the face of satiety signals from POMC neurons; (iii) the opposing effects on food intake are associated with distinct neuronal projection and activation patterns of adult hypothalamic POMC neurons versus Arc neurons derived from POMC-expressing lineages; and (iv) the increased food intake following the activation of orexigenic neurons derived from POMC-expressing progenitors engages an extensive neural network that involves the endogenous opioid system. Together, these findings shed further light on the dynamic balance between two peptidergic systems in the moment-to-moment regulation of feeding behavior.

Background: Sarcopenia, the age-related loss of skeletal muscle, is a side effect of androgen deprivation therapy (ADT) for prostate cancer patients. Resident stem cells of skeletal muscle, satellite cells (SCs), are an essential source of progenitors for the growth and regeneration of skeletal muscle. Decreased androgen signaling and deficits in the number and function of SCs are features of aging. Although androgen signaling is known to regulate skeletal muscle, the cellular basis for ADT-induced exacerbation of sarcopenia is unknown. Furthermore, the consequences of androgen deprivation on SC fate in adult skeletal muscle remain largely unexplored. Methods: We examined SC fate in an androgen-deprived environment using immunofluorescence and fluorescence-activated cell sorting (FACS) with SC-specific markers in young castrated mice. To study the effects of androgen deprivation on SC function and skeletal muscle regenerative capacity, young castrated mice were subjected to experimental regenerative paradigms. SC-derived-cell contributions to skeletal muscle maintenance were examined in castrated Pax7 CreER/+ ; ROSA26 mTmG/+ mice. SCs were depleted in Pax7 CreER/+ ; ROSA26 DTA/+ mice to ascertain the consequences of SC ablation in sham and castrated skeletal muscles. Confocal immunofluorescence analysis of neuromuscular junctions (NMJs), and assessment of skeletal muscle physiology, contractile properties, and integrity were conducted. Results: Castration led to SC activation, however this did not result in a decline in SC function or skeletal muscle regenerative capacity. Surprisingly, castration induced SC-dependent maintenance of young skeletal muscle. The functional dependence of skeletal muscles on SCs in young castrated mice was demonstrated by an increase in SC-derived-cell fusion within skeletal muscle fibers. SC depletion was associated with further atrophy and functional decline, as well as the induction of partial innervation and the loss of NMJ-associated myonuclei in skeletal muscles from castrated mice. Conclusion: The maintenance of skeletal muscles in young castrated mice relies on the cellular contributions of SCs. Considering the well-described age-related decline in SCs, the results in this study highlight the need to devise strategies that promote SC maintenance and activity to attenuate or reverse the progression of sarcopenia in elderly androgen-deprived individuals.

The CLARITY technique enables three-dimensional visualization of fluorescent-labeled biomolecules in clarified intact brain samples, affording a unique view of molecular neuroanatomy and neurocircuitry. It is therefore, essential to find the ideal combination for clearing tissue and detecting the fluorescent-labeled signal. This method requires the formation of a formaldehyde-acrylamide fixative-generated hydrogel mesh through which cellular lipid is removed with sodium dodecyl sulfate. Several laboratories have used differential acrylamide and detergent concentrations to achieve better tissue clearing and antibody penetration, but the potential effects upon fluorescent signal retention is largely unknown. In an effort to optimize CLARITY processing procedures we performed quantitative parvalbumin immunofluorescence and lectin-based vasculature staining using either 4 or 8% sodium dodecyl sulfate detergent in combination with different acrylamide formulas in mouse brain slices. Using both confocal and CLARITY-optimized lightsheet microscope-acquired images, we demonstrate that 2% acrylamide monomer combined with 0.0125% bis-acrylamide and cleared with 4% sodium dodecyl sulfate generally provides the most optimal signal visualization amongst various hydrogel monomer concentrations, lipid removal times, and detergent concentrations.

Tissue infiltration and elevated peripheral circulation of granulocytic myeloid-derived cells is associated with poor outcomes in prostate cancer and other malignancies. Although myeloid-derived cells have the ability to suppress T-cell function, little is known about the direct impact of these innate cells on prostate tumor growth. Here, it is reported that granulocytic myeloid-derived suppressor cells (MDSC) are the predominant tumor-infiltrating cells in prostate cancer xenografts established in athymic nude mice. MDSCs significantly increased in number in the peripheral circulation as a function of xenograft growth and were successfully depleted in vivo by Gr-1 antibody treatment. Importantly, MDSC depletion significantly decreased xenograft growth. We hypothesized that granulocytic MDSCs might exert their protumorigenic actions in part through neutrophil elastase (ELANE), a serine protease released upon granulocyte activation. Indeed, it was determined that NE is expressed by infiltrating immune cells and is enzymatically active in prostate cancer xenografts and in prostate tumors of prostate-specific Pten-null mice. Importantly, treatment with sivelestat, a small-molecule inhibitor specific for NE, significantly decreased xenograft growth, recapitulating the phenotype of Gr-1 MDSC depletion. Mechanistically, NE activated MAPK signaling and induced MAPK-dependent transcription of the proliferative gene cFOS in prostate cancer cells. Functionally, NE stimulated proliferation, migration, and invasion of prostate cancer cells in vitro. IHC on human prostate cancer clinical biopsies revealed coexpression of NE and infiltrating CD33+ MDSCs. Implications: This report suggests that MDSCs and NE are physiologically important mediators of prostate cancer progression and may serve as potential biomarkers and therapeutic targets. Mol Cancer Res; 15(9); 1138–52. ©2017 AACR.

OBJECTIVE Patients with type 1 diabetes (T1D) with impaired renal function are at increased risk for end-stage renal disease (ESRD). Although the rate of progression varies, determinants and mechanisms of this variation are unknown. RESEARCH DESIGN AND METHODS We examined serum metabolomic profiles associated with variation in renal function decline in participants with T1D (the Joslin Kidney Study prospective cohort). One hundred fifty-eight patients with proteinuria and chronic kidney disease stage 3 were followed for a median of 11 years to determine estimated glomerular filtration rate slopes from serial measurements of serum creatinine and to ascertain time to onset of ESRD. Baseline serum samples were subjected to global metabolomic profiling. RESULTS One hundred ten amino acids and purine and pyrimidine metabolites were detected in at least 80% of participants. Serum levels of seven modified metabolites (C-glycosyltryptophan, pseudouridine, O-sulfotyrosine, N-acetylthreonine, N-acetylserine, N6-carbamoylthreonyladenosine, and N6-acetyllysine) were associated with renal function decline and time to ESRD (P < 0.001) independent of the relevant clinical covariates. The significant metabolites correlated with one another and with the indices of tubular injury. CONCLUSIONS This prospective cohort study in participants with T1D, proteinuria, and impaired renal function at baseline demonstrated that patients with increased circulating levels of certain modified metabolites experience faster renal function decline, leading to ESRD. Whether some of these candidate metabolites are risk factors or just prognostic biomarkers of progression to ESRD in T1D needs to be determined.

Hintergrund: Die Klimakrise ist eine potenziell existenzielle Bedrohung der Menschheit und seine gesundheitlichen Folgen sind eine zunehmend wichtige Herausforderung für die Hausarztpraxis. Die Folgen des Klimawandels zeigen sich bereits und werden mit anhaltenden und zunehmenden Hitzeperioden,[zum vollständigen Text gelangen Sie über die oben angegebene URL], 53. Kongress für Allgemeinmedizin und Familienmedizin

Transcript labelling in intact tissues using in situ hybridization chain reaction has potential to provide vital spatiotemporal information for molecular characterization of heterogeneous neuronal populations. However, it remains relatively unexplored in fresh-frozen brain which provides more flexible utilization than perfused tissue. In the present study, we optimized the combination of in situ hybridization chain reaction in fresh-frozen rodent brains and then evaluated the uniformity of neuronal labelling between two clearing methods, CLARITY and iDISCO+. We found that CLARITY yielded higher signal-to-noise ratios but more limited imaging depth and required longer clearing times, whereas, iDISCO+ resulted in better tissue clearing, greater imaging depth and a more uniform labelling of larger samples. Based on these results, we used iDISCO+-cleared fresh-frozen rodent brains to further validate this combination and map the expression of a few genes of interest pertaining to mood disorders. We then examined the potential of in situ hybridization chain reaction to label transcripts in cleared postmortem human brain tissues. The combination failed to produce adequate mRNA labelling in postmortem human cortical slices but produced visually adequate labeling in the cerebellum tissues. We next, investigated the multiplexing ability of in situ hybridization chain reaction in cleared tissues which revealed inconsistent fluorescence output depending upon the fluorophore conjugated to the hairpins. Finally, we applied our optimized protocol to assess the effect of glucocorticoid receptor overexpression on basal somatostatin expression in the mouse cortex. The constitutive glucocorticoid receptor overexpression resulted in lower number density of somatostatin-expressing neurons compared to wild type. Overall, the combination of in situ hybridization chain reaction with clearing methods especially iDISCO+ may find broad application in the transcript analysis in rodent studies but its limited use in postmortem human tissues can be improved by further optimizations.

Transcript labeling in intact tissues using in situ hybridization chain reaction has potential to provide vital spatiotemporal information for molecular characterization of heterogeneous neuronal populations. However, large tissue labeling in non-perfused or fresh-frozen rodent and postmortem human samples, which provide more flexible utilization than perfused tissues, is largely unexplored. In the present study, we optimized the combination of in situ hybridization chain reaction in fresh-frozen rodent brains and then evaluated the uniformity of neuronal labeling between two clearing methods, CLARITY and iDISCO(+). We found that CLARITY yielded higher signal-to-noise ratios but more limited imaging depth and required longer clearing times, whereas, iDISCO(+) resulted in better tissue clearing, greater imaging depth and a more uniform labeling of larger samples. Based on these results, we used iDISCO(+)-cleared fresh-frozen rodent brains to further validate this combination and map the expression of a few genes of interest pertaining to mood disorders. We then examined the potential of in situ hybridization chain reaction to label transcripts in cleared postmortem human brain tissues. The combination failed to produce adequate mRNA labeling in postmortem human cortical slices but produced visually adequate labeling in the cerebellum tissues. We next, investigated the multiplexing ability of in situ hybridization chain reaction in cleared tissues which revealed inconsistent fluorescence output depending upon the fluorophore conjugated to the hairpins. Finally, we applied our optimized protocol to assess the effect of glucocorticoid receptor overexpression on basal somatostatin expression in the mouse cortex. The constitutive glucocorticoid receptor overexpression resulted in lower number density of somatostatin-expressing neurons compared to wild type. Overall, the combination of in situ hybridization chain reaction with clearing methods, especially iDISCO(+), may find broad application in the transcript analysis in rodent studies, but its limited use in postmortem human tissues can be improved by further optimizations.

Background: MicroRNAs (miRNAs) are involved in gene regulation and play important roles in the etiology of various diseases including diabetic kidney disease. The objective of this study was to determine miRNAs profile and their target proteins associated with risk of progression to end-stage renal disease (ESRD) in diabetes. Methods: Using HTG edge sequence platform, 2,083 miRNAs were measured in baseline plasma specimens of patients with renal impairment (CKD stage 3 and 4). We also measured 1,029 protein levels using SomaScan proteomics platform to assess the target proteins of candidate miRNAs. We studied 375 patients with diabetes: 239 patients with type 1 diabetes as our exploratory panel and 136 patients with type 2 diabetes as our replication panel. Results: We identified 19 circulating miRNAs that were strongly associated with progression to ESRD in both T1D and T2D. Among them, 4 representative miRNAs were selected. In pathway analysis, Axon guidance (AG) was the most significant pathway enriched by genes targeted by the representative miRNAs. Our proteomics platform included 41 AG proteins and 6 of them were identified to be associated with ESRD in T1D and T2D. Mediation analysis revealed that 10-83% of the miRNAs effect on ESRD is mediated through axon guidance proteins, namely 2 Ephrin ligands and 4 Ephrin receptors. Conclusions: Our results suggest that certain miRNAs play a significant etiological role in progression of diabetic kidney disease to ESRD mediated by axon guidance proteins. These miRNAs and AG proteins have the potential to be used as novel therapeutic targets and prognostic biomarkers. Disclosure E. Satake: None. J. Skupien: None. Z.I. Md Dom: None. H. Kobayashi: None. K. Ihara: None. M.G. Pezzolesi: None. M. Niewczas: None. A. Krolewski: None. Funding National Institutes of Health; Novo Nordisk Foundation; Sunstar Foundation

Seven AGP proteins associate with ESKD in Caucasians with diabetes. We explored the association of these proteins with ESKD and kidney lesions in Pima Indians with type 2 diabetes. EPHA1, EPHA2, EPHB6, EFNA4, EFNA5, UNC5Cand UNC5D were measured in serum of 162 Pimason a SOMAscan platform. Hazard ratio (HR) for ESKD for a 1 SD increase of each protein was computed by Cox regression after adjustment for age, sex, A1c, iothalamate glomerular filtration rate (GFR), and albumin:creatinine ratio (ACR). Serum samples obtained a median of 2.5 years later were measured in a subset of 67 Pimas who underwent kidney biopsy. Relationships between changes in protein concentrations and kidney lesions were explored by Spearman correlations. Baseline, mean age was 45±10 years, diabetes duration 15±6 years, GFR 155±53 ml/minute, and median ACR 54 (13-250) mg/g. During median follow-up of 12.5 years, 54 participants developed ESKD. Higher concentrations of 6 proteins (UNC5C HR1.51 [95CI 1.07-2.13]; EPHB6 1.67 [1.15-2.41]; EFNA4 1.72 [1.29-2.31]; UNC5D 1.73 [1.12-2.69] EFNA5 1.99 [1.36-2.91] and EPHA2 2.15[1.46-3.17]) were associated with ESKD. Increasing concentrations of these proteins were associated with structural lesions of diabetes (Figure). Proteins related to cell surface molecules of the Ephrin family were associated with kidney lesions and with progression to ESKD in Pima Indians with type 2 diabetes. Disclosure P. Saulnier: None. E. Satake: None. H.C. Looker: None. J. Skupien: None. A. Krolewski: None. R. Nelson: None. Funding American Diabetes Association (1-08-CR-42 to R.N.)

Purpose: Biomarkers are needed to stratify patients with stage II–III melanoma for clinical trials of adjuvant therapy because, while immunotherapy is protective, it also confers the risk of severe toxicity. We previously defined and validated a 53-immune gene melanoma immune profile (MIP) predictive both of distant metastatic recurrence and of disease-specific survival (DSS). Here, we test MIP on a third independent population. Experimental Design: A retrospective cohort of 78 patients with stage II–III primary melanoma was analyzed using the NanoString assay to measure expression of 53 target genes, and MIP score was calculated. Statistical analysis correlating MIP with DSS, overall survival, distant metastatic recurrence, and distant metastasis-free interval was performed using ROC curves, Kaplan–Meier curves, and standard univariable and multivariable Cox proportional hazards models. Results: MIP significantly distinguished patients with distant metastatic recurrence from those without distant metastatic recurrence using ROC curve analysis (AUC = 0.695; P = 0.008). We defined high- and low-risk groups based on the cutoff defined by this ROC curve and find that MIP correlates with both DSS and overall survival by ROC curve analysis (AUC = 0.719; P = 0.004 and AUC = 0.698; P = 0.004, respectively). Univariable Cox regression reveals that a high-risk MIP score correlates with DSS (P = 0.015; HR = 3.2). Conclusions: MIP identifies patients with low risk of death from melanoma and may constitute a clinical tool to stratify patients with stage II–III melanoma for enrollment in clinical trials.

BACKGROUND: Although diabetic kidney disease demonstrates both familial clustering and single nucleotide polymorphism heritability, the specific genetic factors influencing risk remain largely unknown.METHODS: To identify genetic variants predisposing to diabetic kidney disease, we performed genome-wide association study (GWAS) analyses. Through collaboration with the Diabetes Nephropathy Collaborative Research Initiative, we assembled a large collection of type 1 diabetes cohorts with harmonized diabetic kidney disease phenotypes. We used a spectrum of ten diabetic kidney disease definitions based on albuminuria and renal function.RESULTS: Our GWAS meta-analysis included association results for up to 19,406 individuals of European descent with type 1 diabetes. We identified 16 genome-wide significant risk loci. The variant with the strongest association (rs55703767) is a common missense mutation in the collagen type IV alpha 3 chain (COL4A3) gene, which encodes a major structural component of the glomerular basement membrane (GBM). Mutations in COL4A3 are implicated in heritable nephropathies, including the progressive inherited nephropathy Alport syndrome. The rs55703767 minor allele (Asp326Tyr) is protective against several definitions of diabetic kidney disease, including albuminuria and ESKD, and demonstrated a significant association with GBM width; protective allele carriers had thinner GBM before any signs of kidney disease, and its effect was dependent on glycemia. Three other loci are in or near genes with known or suggestive involvement in this condition (BMP7) or renal biology (COLEC11 and DDR1).CONCLUSIONS: The 16 diabetic kidney disease-associated loci may provide novel insights into the pathogenesis of this condition and help identify potential biologic targets for prevention and treatment.

OBJECTIVE Patients with type 1 diabetes and diabetic nephropathy are targets for intervention to reduce high risk of end-stage renal disease (ESRD) and deaths. This study compares risks of these outcomes in four international cohorts. RESEARCH DESIGN AND METHODS In the 1990s and early 2000s, Caucasian patients with type 1 diabetes with persistent macroalbuminuria in chronic kidney disease stages 1–3 were identified in the Joslin Clinic (U.S., 432), Finnish Diabetic Nephropathy Study (FinnDiane) (Finland, 486), Steno Diabetes Center Copenhagen (Denmark, 368), and INSERM (France, 232) and were followed for 3–18 years with annual creatinine measurements to ascertain ESRD and deaths unrelated to ESRD. RESULTS During 15,685 patient-years, 505 ESRD cases (rate 32/1,000 patient-years) and 228 deaths unrelated to ESRD (rate 14/1,000 patient-years) occurred. Risk of ESRD was associated with male sex; younger age; lower estimated glomerular filtration rate (eGFR); higher albumin/creatinine ratio, HbA1c, and systolic blood pressure; and smoking. Risk of death unrelated to ESRD was associated with older age, smoking, and higher baseline eGFR. In adjusted analysis, ESRD risk was highest in Joslin versus reference FinnDiane (hazard ratio [HR] 1.44, P = 0.003) and lowest in Steno (HR 0.54, P < 0.001). Differences in eGFR slopes paralleled risk of ESRD. Mortality unrelated to ESRD was lowest in Joslin (HR 0.68, P = 0.003 vs. the other cohorts). Competing risk did not explain international differences in the outcomes. CONCLUSIONS Despite almost universal renoprotective treatment, progression to ESRD and mortality in patients with type 1 diabetes with advanced nephropathy are still very high and differ among countries. Finding causes of these differences may help reduce risk of these outcomes.

Chronic inflammation is postulated to be involved in development of end stage renal disease (ESRD) in diabetes, but which specific circulating inflammatory proteins contribute to this risk remains unknown. To study this we examined 194 circulating inflammatory proteins in subjects from three independent cohorts with Type 1 and Type 2 diabetes. In each cohort we identified an extremely robust Kidney Risk Inflammatory Signature (KRIS) consisting of 17 novel proteins enriched for TNF Receptor Superfamily members that was associated with a 10-year risk of ESRD. All these proteins had a systemic, non-kidney source. Our prospective study findings provide strong evidence that KRIS proteins contribute to the inflammatory process underlying ESRD development in both types of diabetes. These proteins may be used as new therapeutic targets, new prognostic tests for high risk of ESRD and as surrogate outcome measures where changes in KRIS levels during intervention can reflect the tested therapy’s effectiveness., One Sentence Summary: Proteomic profiling of circulating proteins in subjects from three independent cohorts with type 1 and type 2 diabetes, identified an extremely robust inflammatory signature, consisting of 17 proteins enriched for TNF Receptor Superfamily members that was associated with a 10-year risk of end-stage renal disease.

Background: It has been reported that microRNAs (miRNAs) play an important role in the pathogenesis of diabetic complications. We aimed to search for circulating miRNA that were associated with hyperglycemia in type 2 diabetes and examine their effects on renal function decline. Methods: Using the next-generation sequencing-based HTG EdgeSeq miRNA platform, a total of 2,083 miRNAswere measured in baseline plasma specimens obtained from73 subjects with type 2 diabetes (T2D) and normal renal function (discovery panel) and 136 subjects with T2D and impaired renal function (replication panel). Subjects in both panels were followed for 6–12 years to determine eGFR decline. Results: We identified 11 candidate miRNAsthat were strongly associated with elevated levels of glycated hemoglobin (HbA1c) in both screening and replication panels. Using bioinformatics analyses, we found that the candidate miRNAs targeted proteins of 6 pathways (the Ras signaling pathway, Signaling pathways regulating pluripotency of stem cells, the MAPK pathway, Glutamatergic synapse, the Rap 1 signaling pathway, and the AMPK signaling pathway). Importantly, 4 of these 11 miRNAs were significantly associated with risk of renal function decline. Conclusion: There were few previous reports about the association between circulating miRNAs, hyperglycemia, and diabetic kidney disease in T2D. The present study comprehensively examined and identified hyperglycemia-regulated miRNAs in human samples. Our finding are novel in that circulatingmiRNAsregulated by hyperglycemia are associated with risk of eGFR decline in T2D.

First line treatment for recurrent and metastatic prostate cancer is androgen deprivation therapy (ADT). Use of ADT has been increasing in frequency and duration, such that side effects increasingly impact patient quality of life. One of the most significant side effects of ADT is sarcopenia, which leads to a loss of skeletal muscle mass and function, resulting in a clinical disability syndrome known as obese frailty. Using aged mice, we developed a mouse model of ADT-induced sarcopenia that closely resembles the phenotype seen in patients, including loss of skeletal muscle strength, reduced lean muscle mass, and increased adipose tissue. Sarcopenia onset occurred about 6 weeks after castration and was blocked by a soluble receptor (ActRIIB-Fc) that binds multiple TGFβ superfamily members, including myostatin, growth differentiation factor 11, activin A, activin B, and activin AB. Analysis of ligand expression in both gastrocnemius and triceps brachii muscles demonstrates that each of these proteins is induced in response to ADT, in 1 of 3 temporal patterns. Specifically, activin A and activin AB levels increase and decline before onset of strength loss at 6 weeks after castration, and myostatin levels increase coincident with the onset of strength loss and then decline. In contrast, activin B and growth differentiation factor 11 levels increase after the onset of strength loss, 8-10 weeks after castration. The observed patterns of ligand induction may represent differential contributions to the development and/or maintenance of sarcopenia. We hypothesize that some or all of these ligands are targets for therapy to ameliorate ADT-induced sarcopenia in prostate cancer patients.

Obesity is a persistent and pervasive problem, particularly in industrialized nations. It has come to be appreciated that the metabolic health of an individual can influence brain function and subsequent behavioral patterns. To examine the relationship between metabolic phenotype and central systems that regulate behavior, we tested rats with divergent metabolic phenotypes (Low Capacity Runner: LCR vs. High Capacity Runner: HCR) for behavioral responses to the conflict between hunger and environmental novelty using the novelty suppressed feeding (NSF) paradigm. Additionally, we measured expression of mRNA, for peptides involved in energy management, in response to fasting. Following a 24-h fast, LCR rats showed lower latencies to begin eating in a novel environment compared to HCR rats. A 48-h fast equilibrated the latency to begin eating in the novel environment. A 24-h fast differentially affected expression of cocaine-amphetamine regulated transcript (CART) mRNA in the nucleus accumbens (NAc), where 24-h of fasting reduced CART mRNA in LCR rats. Bilateral microinjections of CART 55–102 peptide into the NAc increased the latency to begin eating in the NSF paradigm following a 24-h fast in LCR rats. These results indicate that metabolic phenotype influences how animals cope with the conflict between hunger and novelty, and that these differences are at least partially mediated by CART signaling in the NAc. For individuals with poor metabolic health who have to navigate food-rich and stressful environments, changes in central systems that mediate conflicting drives may feed into the rates of obesity and exacerbate the difficulty individuals have in maintaining weight loss.

Progressively decreasing glomerular filtration rate (GFR), or renal decline, is seen in patients with type 1 diabetes (T1D) and normoalbuminuria or microalbuminuria. Here we examined the associations of kidney injury molecule-1 (KIM-1) in plasma and urine with the risk of renal decline and determine whether those associations are independent of markers of glomerular damage. The study group comprised patients with T1D from the 2nd Joslin Kidney Study of which 259 had normoalbuminuria and 203 had microalbuminuria. Serial measurements over 4 to 10 years of follow-up (median 8 years) of serum creatinine and cystatin C were used jointly to estimate eGFRcr-cys slopes and time of onset of CKD stage 3 or higher. Baseline urinary excretion of IgG2 and albumin were used as markers of glomerular damage, and urinary excretion of KIM-1 and its plasma concentration were used as markers of proximal tubular damage. All patients had normal renal function at baseline. During follow-up, renal decline (eGFRcr-cys loss 3.3% or more per year) developed in 96 patients and 62 progressed to CKD stage 3. For both outcomes, the risk rose with increasing baseline levels of plasma KIM-1. In multivariable models, elevated baseline plasma KIM-1 was strongly associated with risk of early progressive renal decline, regardless of baseline clinical characteristics, serum TNFR1 or markers of glomerular damage. Thus, damage to proximal tubules may play an independent role in the development of early progressive renal decline in non-proteinuric patients with T1D.

Elevated serum tumor necrosis factor receptor 1 (TNFR1) and 2 (TNFR2) concentrations are strongly associated with increased risk of end-stage renal disease in type 2 diabetes. However, little is known about the early glomerular structural lesions that develop in patients when these markers are elevated. Here, we examined the relationships between TNFRs and glomerular structure in 83 American Indians with type 2 diabetes. Serum TNFRs and glomerular filtration rate (GFR, iothalamate) were measured during a research exam performed within a median of 0.9 months from a percutaneous kidney biopsy. Associations of TNFRs with glomerular structural variables were quantified by Spearman's correlations and by multivariable linear regression after adjustment for age, gender, diabetes duration, hemoglobin A1c, body mass index, and mean arterial pressure. The baseline mean age was 46 years, median GFR 130 ml/min, median albumin/creatinine ratio 26 mg/g, median TNFR1 1500 pg/ml, and median TNFR2 3284 pg/ml. After multivariable adjustment, TNFR1 and TNFR2 significantly correlated inversely with the percentage of endothelial cell fenestration and the total filtration surface per glomerulus. There were significant positive correlations with mesangial fractional volume, glomerular basement membrane width, podocyte foot process width, and percentage of global glomerular sclerosis. Thus, TNFRs may be involved in the pathogenesis of early glomerular lesions in diabetic nephropathy.

Diabetic kidney disease (DKD) is a heritable but poorly understood complication of diabetes. To identify genetic variants predisposing to DKD, we performed genome-wide association analyses in 19,406 individuals with type 1 diabetes (T1D) using a spectrum of DKD definitions basedon albuminuria and renal function. We identified 16 genome-wide significant loci. The variant with the strongest association (rs55703767) is a common missense mutation in the collagen type IV alpha 3 chain(COL4A3)gene, which encodes a major structural component of the glomerular basement membrane (GBM) implicated in heritable nephropathies. The rs55703767 minor allele (Asp326Tyr) is protective against several definitions of DKD, including albuminuria and end-stage renal disease. Three other loci are in or near genes with known or suggestive involvement in DKD(BMP7)or renal biology (COLEC11andDDR1). The 16 DKD-associated loci provide novel insights into the pathogenesis of DKD, identifying potential biological targets for prevention and treatment.

Aging is the biggest risk factor for several neurodegenerative diseases. Parabiosis experiments have established that old mouse brains are improved by exposure to young mouse blood. Previously, our lab showed that delivery of Growth Differentiation Factor 11 (GDF11) to the bloodstream increases the number of neural stem cells and positively affects vasculature in the subventricular zone of old mice. Our new study demonstrates that GDF11 enhances hippocampal neurogenesis, improves vasculature and increases markers of neuronal activity and plasticity in the hippocampus and cortex of old mice. Our experiments also demonstrate that systemically delivered GDF11, rather than crossing the blood brain barrier, exerts at least some of its effects by acting on brain endothelial cells. Thus, by targeting the cerebral vasculature, GDF11 has a very different mechanism from that of previously studied circulating factors acting to improve central nervous system (CNS) function without entering the CNS.

A scientific session entitled “New Frontiers: Approaches to Understand the Mechanistic Basis of Renal Toxicity” focused on novel biomarkers to monitor kidney injury both preclinically and clinically, as well as providing mechanistic insight of the induced injury. Further, the role and impact of kidney membrane transporters in drug-induced kidney toxicity provided additional considerations when understanding kidney injury and the complex role of drug transporters in either sensitivity or resistance to drug-induced injury. The onset of nephropathy in diabetic patients was also presented, focusing on the quest to discover novel biomarkers that would differentiate diabetic populations more susceptible to nephropathy and renal failure. The session highlighted exciting new research areas and novel biomarkers that will enhance our understanding of kidney injury and provide tools for ensuring patient safety clinically.

We conducted an untargeted proteomic profiling of circulating inflammatory proteins using SOMASCAN platform to find those that predicted 10-year ESRD risk in two cohort study. Subjects with Type 1 Diabetes (T1D) served as a discovery cohort and a T2D cohort was used in validation. We identified a kidney risk inflammatory signature (KRIS) that robustly predicted progression to ESRD. The signature comprised 17 proteins out of 194 examined. KRIS was enriched for members of TNFR Superfamily (p=0.007) including TNFR1, TNFR2, TNFRSF19, TNFRSF19L, TNFRSF21 and TNFRSF27. Other KRIS proteins included IL15RA, IL17F, DAF, CLM6, TNFSF15, CCL14, CCL15, CSF1, TIMD3, IL1R1 and IL18R1. Hazard ratio (95% CI) for the top KRIS protein, TNFR1 was 3.6 (2.8, 4.6), p < 10-25. KRIS was neither enriched in receptors for other cytokines nor in cytokine ligands. Pathway analyses pointed to candidate therapeutic targets and aligned them by ranks. Eight proteins are currently targeted with compounds used for other clinical indications. Kidney tissue expression analysis and urinary proteomics suggested a systemic source of KRIS proteins. In summary, our study identifies and validates a powerful protein signature enriched in TNFRSF members associated with 10-year ESRD risk in diabetes. These findings should inform future drug development strategies. Disclosure M. Niewczas: None. J.K. Skupien: None. V. Nair: None. A. Smiles: None. J. Park: None. E. Satake: None. C.A. Simeone: None. J. Tsay: None. W. Ju: None. M. Kretzler: Research Support; Self; AstraZeneca, Novo Nordisk Inc., Boehringer Ingelheim GmbH, National Institute of Diabetes and Digestive and Kidney Diseases, Eli Lilly and Company, Gilead Sciences, Inc. K. Susztak: Research Support; Self; Regeneron Pharmaceuticals, Inc., Boehringer Ingelheim Pharmaceuticals, Inc., GlaxoSmithKline plc., Merck & Co., Inc., Eli Lilly and Company, Ono Pharmaceutical Co., Ltd.. A. Krolewski: None.

We have reported that up-regulation of PKM2 in renal glomeruli isolated from patients with more than 50 years of type 1 diabetes (Joslin Medalist Study) correlated with preservation of renal function even in the presence of chronic hyperglycemia. Further, pharmacological activation of PKM2 in T1D mice and podocyte-specific deletion of PKM2 in cultured cells and in vivo strongly showed that PKM2 activation protects renal glomeruli from hyperglycemia/diabetes induced damage (Nat. Med 2017). To confirm these findings in type 1 and type 2 diabetes, we performed the following: 1. We showed the protein expressions of glycolytic (PKM1, PKM2, ENO1) and mitochondrial (MTCO2) enzymes to be significantly elevated in glomeruli of post mortem kidneys in people with T2D without DN compared to those with DN, or nondiabetic controls. 2. Plasma PKM2 levels (SomaLogic aptamer assay) in the Medalists (n=180) correlated with albumin-to-creatinine ratio (ACR;p=0.03) and eGFR (p=0.0002) at baseline. In a replication cohort (n=219) of patients with T1D in the longitudinal Joslin Kidney Study (JKS) baseline plasma PKM2 also correlated with ACR (p=0.0001). High baseline plasma PKM2 in JKS was independently associated with a slower decline in eGFR (p=0.004) and a slower progression to end-stage renal disease (p=0.0002). 3. TEPP-46, a PKM2 activator, prevented diabetes induced increases in mRNA expression of pro-fibrotic (Col3a1, Fn1, TGFb1) and inflammatory (Ccl2) genes, and enhanced key mitochondrial protein (Ppargc1a) in uninephrectomized T2D db/db mice. No differences in ACR were observed between TEPP-46 treated and untreated diabetic mice. These findings support the hypothesis that PKM2 activation may protect or even reverse the initiation and progression of diabetic glomerular dysfunction and pathology in both T1D and T2D, suggesting that activating PKM2 could be a clinically effective therapeutic target to stop the progression of DN. Disclosure D. Gordin: None. T. Shinjo: None. R. St-Louis: None. W. Qi: None. L.J. Tinsley: None. D.M. Pober: None. M.J. Brissett: None. M. Niewczas: None. J. Dreyfuss: None. H. Pan: None. T. Sadowski: Employee; Self; Sanofi. A. Kannt: Employee; Self; Sanofi. Stock/Shareholder; Self; Sanofi. H. Shah: None. H.A. Keenan: Research Support; Self; Sanofi. Employee; Self; Sanofi Genzyme. A. Krolewski: None. G.L. King: Research Support; Self; Sanofi-Aventis.

To identify determinants of early progressive renal decline in type 2 diabetes a range of markers was studied in 1032 patients enrolled into the 2nd Joslin Kidney Study. eGFR slopes estimated from serial measurements of serum creatinine during 5-12 years of follow-up were used to define early renal decline. At enrollment, all patients had normal eGFR, 58% had normoalbuminuria and 42% had albuminuria. Early renal decline developed in 6% and in 18% patients, respectively. As determinants, we examined baseline values of clinical characteristics, circulating markers: TNFR1, KIM-1, and FGF23, and urinary markers: albumin, KIM-1, NGAL, MCP-1, EGF (all normalized to urinary creatinine) and the ratio of EGF to MCP-1. In univariate analysis, all plasma and urinary markers were significantly associated with risk of early renal decline. When analyzed together, systolic blood pressure, TNFR1, KIM-1, the albumin to creatinine ratio, and the EGF/MCP-1 ratio remained significant with the latter having the strongest effect. Integration of these markers into a multi-marker prognostic test resulted in a significant improvement of discriminatory performance of risk prediction of early renal decline, compared with the albumin to creatinine ratio and systolic blood pressure alone. However, the positive predictive value was only 50% in albuminuric patients. Thus, markers in plasma and urine indicate that the early progressive renal decline in Type 2 diabetes has multiple determinants with strong evidence for involvement of tubular damage. However, new, more informative markers are needed to develop a better prognostic test for such decline that can be used in a clinical setting.

In the retina, dopamine is a key molecule for daytime vision. Dopamine is released by retinal dopaminergic amacrine cells and transmits signaling either by conventional synaptic or by volume transmission. By means of volume transmission, dopamine modulates all layers of retinal neurons; however, it is not well understood how dopamine modulates visual signaling pathways in bipolar cells. Here we analyzed Drd1a-tdTomato BAC transgenic mice and found that the dopamine D1 receptor (D1R) is expressed in retinal bipolar cells in a type-dependent manner. Strong tdTomato fluorescence was detected in the inner nuclear layer and localized to type 1, 3b, and 4 OFF bipolar cells and type 5-2, XBC, 6, and 7 ON bipolar cells. In contrast, type 2, 3a, 5-1, 9, and rod bipolar cells did not express Drd1a-tdTomato. Other interneurons were also found to express tdTomato including horizontal cells and a subset (25%) of AII amacrine cells. Diverse visual processing pathways, such as color or motion-coded pathways, are thought to be initiated in retinal bipolar cells. Our results indicate that dopamine sculpts bipolar cell performance in a type-dependent manner to facilitate daytime vision. J. Comp. Neurol. 524:2059-2079, 2016. © 2015 Wiley Periodicals, Inc.

The dramatic responses tumors display to targeted therapies are limited by acquired or pre-existing mechanisms of therapy resistance. We recently discovered that androgen receptor blockade by the anti-androgen enzalutamide paradoxically enhanced metastasis and that these pro-metastatic effects were mediated by the chemoattractant CCL2. CCL2 is regulated by TNF, which is negatively regulated by androgen signaling. Thus, we asked if TNF mediates the pro-metastatic effects of enzalutamide. We found that androgen withdrawal or enzalutamide induced TNF mRNA and protein secretion in castration resistant prostate cancer (C4-2) cells, but not in macrophage-like (THP1) or myofibroblast-like (WPMY1) cells. Androgen deprivation therapy (ADT) induced autocrine CCL2 expression in C4-2 (as well as a murine CRPC cell line), while exogenous TNF induced CCL2 in THP1 and WPMY1. TNF was most potent in myofibroblast cultures, suggesting ADT induces CCL2 via paracrine interactions within the tumor microenvironment. A soluble TNF receptor (etanercept) blocked enzalutamide-induced CCL2 protein secretion and mRNA, implying dependence on secreted TNF. A small molecule inhibitor of CCR2 (the CCL2 receptor) significantly reduced TNF induced migration, while etanercept inhibited enzalutamide-induced migration and invasion of C4-2. Analysis of human prostate cancers suggests that a TNF-CCL2 paracrine loop is induced in response to ADT and might account for some forms of prostate cancer therapy resistance.

On the basis of extensive studies in Joslin Clinic patients over 25 years, we propose a new model of diabetic nephropathy in type 1 diabetes. In this model, the predominant clinical feature of both early and late stages of diabetic nephropathy is progressive renal decline, not albuminuria. Progressive renal decline (estimated glomerular filtration rate loss >3.5 mL/min/year) is a unidirectional process that develops while patients have normal renal function. It progresses at an almost steady rate until end-stage renal disease is reached, albeit at widely differing rates among individuals. Progressive renal decline precedes the onset of microalbuminuria, and as it continues, it increases the risk of proteinuria. Therefore, study groups ascertained for microalbuminuria/proteinuria are enriched for patients with renal decline (decliners). We found prevalences of decliners in 10%, 32%, and 50% of patients with normoalbuminuria, microalbuminuria, and proteinuria, respectively. Whether the initial lesion of progressive renal decline is in the glomerulus, tubule, interstitium, or vasculature is unknown. Similarly unclear are the initiating mechanism and the driver of progression. No animal model mimics progressive renal decline, so etiological studies must be conducted in humans with diabetes. Prospective studies searching for biomarkers predictive of the onset and rate of progression of renal decline have already yielded positive findings that will help to develop not only accurate methods for early diagnosis but also new therapeutic approaches. Detecting in advance which patients will have rapid, moderate, or minimal rates of progression to end-stage renal disease will be the foundation for developing personalized methods of prevention and treatment of progressive renal decline in type 1 diabetes.

// Hongjun Xie 1, 2, * , Lei Li 1, 2, * , Guodong Zhu 1, 2 , Qiang Dang 1, 2 , Zhenkun Ma 1 , Dalin He 1 , Luke Chang 1 , Wenbing Song 1, 2 , Hong-Chiang Chang 2 , John J. Krolewski 3 , Kent L. Nastiuk 3 , Shuyuan Yeh 2 , Chawnshang Chang 2, 4 1 Sex Hormone Research Center, Department of Urology, The First Affiliated Hospital, Xi’an Jiaotong University, Xi’an, China 2 George Whipple Lab for Cancer Research, Departments of Pathology and Urology, and The Wilmot Cancer Center, University of Rochester Medical Center, Rochester, New York, USA 3 Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, New York, USA 4 Sex Hormone Research Center, China Medical University/Hospital, Taichung, Taiwan * These authors have contributed equally to this work Correspondence to: Lei Li, e-mail: lilydr@163.com Chawnshang Chang, e-mail: chang@urmc.rochester.edu Keywords: prostate cancer, pre-adipocyte, mirna-301a, androgen receptor Received: February 15, 2015 Accepted: March 15, 2015 Published: April 8, 2015 ABSTRACT High fat dietary intake may increase the risk of prostate cancer (PCa). Pre-adipocytes, one of the basic components in the tumor microenvironment (TME), are capable of differentiating into adipose tissues and play key roles to affect PCa progression. Here we found the pre-adipocytes could be recruited more easily to PCa than its surrounding normal prostate tissue. In vitro co-culture system also confirmed PCa has a better capacity than normal prostate to recruit pre-adipocytes. The consequences of recruiting more pre-adipocytes may then increase PCa cell invasion. Mechanism dissection revealed infiltrating pre-adipocytes might function through down-regulation of the androgen receptor (AR) via modulation of miR-301a, and then increase PCa cell invasion via induction of TGF-β1/Smad/MMP9 signals. The mouse model with orthotopically xenografted PCa CWR22Rv1 cells with pre-adipocytes also confirmed that infiltrating pre-adipocytes could increase PCa cell invasion via suppressing AR signaling. Together, our results reveal a new mechanism showing pre-adipocytes in the prostate TME can be recruited to PCa to increase PCa metastasis via modulation of the miR-301a/AR/TGF-β1/Smad/MMP9 signals. Targeting this newly identified signaling may help us to better inhibit PCa metastasis.

We investigated whether circulating TGF-β1–regulated miRNAs detectable in plasma are associated with the risk of rapid progression to end-stage renal disease (ESRD) in a cohort of proteinuric patients with type 1 diabetes (T1D) and normal eGFR. Plasma specimens obtained at entry to the study were examined in two prospective subgroups that were followed for 7–20 years (rapid progressors and nonprogressors), as well as a reference panel of normoalbuminuric T1D patients. Of the five miRNAs examined in this study, let-7c-5p and miR-29a-3p were significantly associated with protection against rapid progression and let-7b-5p and miR-21-5p were significantly associated with the increased risk of ESRD. In logistic analysis, controlling for HbA1c and other covariates, let-7c-5p and miR-29a-3p were associated with more than a 50% reduction in the risk of rapid progression (P ≤ 0.001), while let-7b-5p and miR-21-5p were associated with a >2.5-fold increase in the risk of ESRD (P ≤ 0.005). This study is the first prospective study to demonstrate that circulating TGF-β1–regulated miRNAs are deregulated early in T1D patients who are at risk for rapid progression to ESRD.